Rather than a mere storage site for triglycerides, it is now understood that adipose tissue (fat) and its cellular units, adipocytes, serve as an endocrine organ that responds to extracellular stimuli. Adiponectin, a multimeric protein secreted by adipocytes, is one of the most abundantly secreted proteins in the body and plays a major role in insulin sensitivity, intermediary metabolism, and vascular inflammation. Recent studies have uncovered that adipose tissue has machinery for acutely secreting adiponectin into the bloodstream; yet without adequate approaches for sensitive detection of adiponectin in small volumes, there is limited understanding of adiponectin secretion dynamics and the response to pharmacological treatments. In particular, there is a clear need for more specific and simple-to-use adiponectin assays. The long-term goal of this research is to understand the molecular mechanisms of acute adiponectin secretion to help uncover its relation to insulin resistance, obesity, and metabolic disease states. The objective of this proposal is to determine the dynamics of acute adiponectin secretion and the effects of commonly used lipid-lowering drugs on these dynamics. To fill this gap in knowledge, homogeneous, sensitive assays (pFRET) will be developed for quantitation of adiponectin multimers from only a few microliters of sample. The simplified readout of the pFRET assays will be combined with microfluidic sampling of primary adipocytes to minimize dilution and measure secretion dynamics at high temporal resolution. The studies will not only eliminate a critical barrier to progress in adipocyt research, but will also address important, unresolved questions on acute adiponectin secretion. Aim 1 of the proposal seeks to develop homogeneous protein assays (pFRET), which are proven for insulin and thrombin detection, for direct fluorescence detection of adiponectin multimers. Aim 2 seeks to determine the dynamics of acute adiponectin secretion, where it is hypothesized that acute (<10 min) adiponectin secretion originates from a reserve pool of vesicles and is not dependent on ER-to-Golgi vesicle trafficking. Microfluidic secretion sampling, combined with the small-volume pFRET, will help interrogate acute secretion. In Aim 3, the PI will investigate the effects of a commonly-used lipid-lowering drug on acute adiponectin secretion, testing the hypothesis that statins increase acutely (<10 min) secreted adiponectin multimers. This proposal is significant because it will overcome a critical barrier to progress in understanding acute adiponectin secretion by filling gaps in the current methodology. The proposal is innovative based on the development and integration of two novel bioanalytical approaches, proximity assays and passive microfluidic sampling, for investigating recently discovered acute adiponectin secretion events that are not accessible without this technology. Preliminary evidence strongly supports the feasibility of these proposals. These findings could better inform the timing of drug administration to the many patients currently taking lipid lowerin drugs and permit future work on uncovering molecular and physiological mechanisms of acute adipokine secretion.